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1.
Rev Bras Parasitol Vet ; 29(3): e003720, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667502

RESUMO

The aim of this study was to report on detection of Toxoplasma gondii DNA in oysters (Crassostrea sp.) in the state of Maranhão. To conduct this study, 200 farmed oysters were acquired in the municipality of Raposa and 100 in Paço do Lumiar; and a further 100 oysters were taken from the natural stock in the municipality of Primeira Cruz. This total of 400 specimens sampled was divided into 80 pools composed of five animals each. The gills and visceral mass of each oyster were removed for DNA extraction (per pool of oysters), using a commercial kit. The nested PCR technique (with the primer SAG-1) was then used to investigate any presence of protozoa. This molecular technique demonstrated the presence of DNA of T. gondii in 2.5% of the pools of oysters (n = 2/80): these oysters were exclusively from farms. The results from this study allow the conclusion that oysters of the genus Crassostrea that are farmed in the state of Maranhão are capable of filtering oocysts of T. gondii and maintaining them in their tissues. They are therefore potential sources of contamination for humans and other animals.


Assuntos
Crassostrea , Toxoplasma , Animais , Aquicultura , Brasil , Crassostrea/parasitologia , DNA de Protozoário/genética , Oocistos/isolamento & purificação , Reação em Cadeia da Polimerase , Toxoplasma/fisiologia
2.
Mikrobiyol Bul ; 54(2): 306-317, 2020 Apr.
Artigo em Turco | MEDLINE | ID: mdl-32723285

RESUMO

Malaria is a life-threatening parasitic disease caused by the parasites belonging to Plasmodium genus. Microscopic examination of Giemsa stained blood smears is accepted as the gold standard diagnostic method. It is recommended to use more than one method in order to strengthen the laboratory diagnosis of malaria which is an important health problem in our country as in the whole world. In this study, it was aimed to compare the results of three different molecular methods and determine which molecular method could be used in the diagnostic algorithm to be applied. DNA was extracted from 280 whole blood sample stored in EDTA tubes using a commercial kit. Three different polymerase chain reaction (PCR) methods were used for the detection of Plasmodium spp. in DNA samples obtained and the results were compared. First, multiplex nested PCR was applied and then in-house real-time PCR (Rt-PCR) which was validated in our laboratory and a commercial Rt-PCR kit were applied. Multiplex nested PCR was accepted as the gold standard and 182 samples that were evaluated as Plasmodium spp. positive and 98 samples that were evaluated as negative were also studied by in-house and commercial Rt-PCR methods. In multiplex nested PCR's first step reaction 1670 base pairs (bp) band was observed in Plasmodium spp. positive samples and 117 bp band was observed in Plasmodium vivax positive samples in the second step reaction. Tm values of P.vivax positive samples were determined as 78-79 in the melting analysis of the in-house Rt-PCR. CT values of the positive samples in in-house Rt-PCR were between 20.03-31.71 and were between 17.26-34.94 in the commercial Rt-PCR. With the in-house Rt-PCR method 180 cases were determined as positive, while with the commercial Rt-PCR method 178 cases were determined as positive. Two samples with the in-house Rt-PCR and 4 samples with the commercial Rt-PCR were considered as false negative. When the sensitivity and specificity of the both methods were calculated, the sensitivity of the in-house Rt-PCR method was 0.98, the specificity was 0.97, the positive predictive value (PPV) was 98%, the negative predictive value (NPV) was 97%, the sensitivity of the commercial Rt-PCR was 0.97, the specificity was 0.95, the PPV was 97%, the NPV was 95%. A high level of agreement (κ: 0.953) was determined between the in-house and the commercial Rt-PCR methods. In order for a test to be accepted as a confirmatory test, its specificity must be high. It was decided that sensitivity and specificity of the in-house Rt-PCR were suitable for using this method in the laboratory diagnosis of Plasmodium species.


Assuntos
Malária , Reação em Cadeia da Polimerase Multiplex , Plasmodium , Reação em Cadeia da Polimerase em Tempo Real , DNA de Protozoário/genética , Humanos , Malária/sangue , Malária/diagnóstico , Malária/parasitologia , Reação em Cadeia da Polimerase Multiplex/normas , Plasmodium/classificação , Plasmodium/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
Rev Bras Parasitol Vet ; 29(2): e003520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520088

RESUMO

Blood samples and swabs from ocular conjunctiva and mouth were obtained from 64 cats. Of 64 serum samples, 19 were positive for Leishmania antibodies by ELISA (29.80%). Eight cats were positive by PCR (12.5%) in swab samples from mouth and/or ocular mucosa. Poor kappa agreement between serological and molecular results (k = 0.16) was obtained. From five positive PCR samples one was L. braziliensis and four were L. infantum. Phylogenetic analysis performed with the five isolates of Leishmania, showed that samples of L. infantum isolated from the cats were phylogenetically close to those isolated from domestic dogs in Brazil, while the L. braziliensis is very similar to the one described in humans in Venezuela. The study demonstrated that, despite high seropositivity for Leishmania in cats living in the study region, poor agreement between serological and molecular results indicate that positive serology is not indicative of Leishmania infection in cats. Parasite DNA can be detected in ocular conjunctiva and oral swabs from cats, indicating that such samples could be used for diagnosis. Results of phylogenetic analyzes show that L. infantum circulating in Brazil is capable of infecting different hosts, demonstrating the parasite's ability to overcome the interspecies barrier.


Assuntos
Doenças do Gato/parasitologia , Leishmania braziliensis/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Doenças do Gato/diagnóstico , Gatos , DNA de Protozoário/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose/diagnóstico , Filogenia , Reação em Cadeia da Polimerase/veterinária
4.
BMC Infect Dis ; 20(1): 319, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32357839

RESUMO

BACKGROUND: Trichomoniasis resulting from Trichomonas vaginalis (T. vaginalis) has been considered as a commonly seen disease with the transmission way of sex. At present, the detection methods of T. vaginalis mainly include wet mount microscopy, culture, PCR, immunofluorescence and ELISA. However, all of these detection methods exist shortcomings. METHODS: In this study, a loop-mediated isothermal amplification (LAMP) assay that targeted the species-specific sequence of adhesion protein 65 (AP65) gene had been conducted to detect T. vaginalis. The optimum reaction system and conditions were optimized in this rapid detection method. RESULTS: The results of sensitivity analysis showed that the LAMP assay targeting the AP65 gene was 1000 times more sensitive than the nested PCR targeting the actin gene commonly used for detection of T. vaginalis, and the detecting limitation of the former was 10 trichomonad. Moreover, the amplification of the target gene AP65 by LAMP assay exhibited high specificity and the product was exclusively from T. vaginalis. The detection technique of LAMP did not exhibit cross-reactivity with the common pathogens of Trichinella spiralis, Toxoplasma gondii, Escherichia coli, Candida albicans, Staphylococcus aureus, Haemophilus. CONCLUSIONS: According to the present study, the LAMP assay with the target of AP65 gene, was suitable for the early diagnosis of T. vaginalis infections. Consequently, the LAMP assay was proposed by the current study as a point-of-care examination and an alternative molecular tool which exhibited the potential value in the treatment, control and prevention of trichomoniasis transmission and relevant complication.


Assuntos
Moléculas de Adesão Celular/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Tricomoníase/diagnóstico , Trichomonas vaginalis/genética , Sequência de Bases/genética , DNA de Protozoário/genética , Diagnóstico Precoce , Feminino , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Tricomoníase/parasitologia , Esfregaço Vaginal
5.
Rev Bras Parasitol Vet ; 29(2): e002420, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428179

RESUMO

Hepatozoon pyramidumi sp. n. is described from the blood of the Egyptian saw-scaled viper, Echis pyramidum, captured from Saudi Arabia. Five out of ten viper specimens examined (50%) were found infected with Hepatozoon pyramidumi sp. n. with parasitaemia level ranged from 20-30%. The infection was restricted only to the erythrocytes. Two morphologically different forms of intraerythrocytic stages were observed; small and mature gamonts. The small ganomt with average size of 10.7 × 3.5 µm. Mature gamont was sausage-shaped with recurved poles measuring 16.3 × 4.2 µm in average size. Infected erythrocytes were hypertrophied; their nuclei were deformed and sometimes displaced from their central position in the normal uninfected cell. Merogonic stages were observed in the lung endothelial cell and the liver parenchyma cells. Mature meront was 17.8 × 13.6 µm and contained banana-shaped merozoites with average size of ~15 × 2 µm. Phylogenetic analysis based on the SSU rDNA sequence clustered Hepatozoon pyramidumi sp. n with previously sequenced Hepatozoon spp., most of them infected reptilian hosts without geographic consideration. The morphological and molecular comparison with closely related species proved the taxonomic uniqueness and novelty of the present form.


Assuntos
Apicomplexa/genética , Apicomplexa/fisiologia , DNA de Protozoário/genética , Viperidae/parasitologia , Animais , Apicomplexa/classificação , DNA Ribossômico/genética , Eritrócitos/parasitologia , Eritrócitos/patologia , Fígado/parasitologia , Fígado/patologia , Pulmão/parasitologia , Pulmão/patologia , Parasitemia/parasitologia , Parasitemia/veterinária , Filogenia , Arábia Saudita , Análise de Sequência de DNA , Viperidae/sangue
6.
Rev Bras Parasitol Vet ; 29(2): e017919, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428181

RESUMO

Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père David's deer. In this study, 137 fecal samples from Père David's deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein (gp60) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père David's deer in this area.


Assuntos
Criptosporidiose/epidemiologia , Cryptosporidium/genética , DNA de Protozoário/genética , Cervos/parasitologia , Epidemiologia Molecular , RNA Ribossômico , Animais , China/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium/isolamento & purificação , Fezes/parasitologia , Genótipo , Filogenia , Prevalência , Análise de Sequência de DNA
7.
Nature ; 582(7810): 104-108, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32427965

RESUMO

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.


Assuntos
Apoptose/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Parasitos/imunologia , Plasmodium falciparum/citologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Aotidae/imunologia , Aotidae/parasitologia , Caspases/metabolismo , Criança , Estudos de Coortes , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Ativação Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Quênia , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Parasitos/citologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Tanzânia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/imunologia , Vacúolos/imunologia
8.
Eur J Protistol ; 74: 125644, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32460119

RESUMO

Three freshwater scuticociliates, Apouronema harbinensis gen. nov. spec. nov., Cyclidium vorax spec. nov., and C. glaucomaMüller, 1773, collected from rivers in Hulan District, Harbin, northeastern China, were investigated using morphological and phylogenetic criteria. Apouronema gen. nov., assigned to the family Uronematidae, is mainly distinguished from the other genera of the family by its paroral membrane extending anteriorly to the middle of membranelle 1. Apouronema harbinensis spec. nov. is defined by body size in vivo about 45-55 × 20-25 µm, buccal field about 70-80% of cell length; 12 or 13 somatic kineties; membranelle 1 having two rows, with 16-18 basal bodies in each kinety; membranelle 2 and membranelle 3 both having two rows each; scutica X-shaped with five pairs of basal bodies. Cyclidium vorax spec. nov. is characterized by the following features: body size 35-40 × 18-20 µm in vivo; 9 or 10 somatic kineties; membranelle 1 having two longitudinal rows, much shorter than M2; M2 triangle-shaped. The phylogenetic analyses show that: (1) Apouronema clustered in the Uronematidae clade, and grouped with genera Uronemita and Uronema; (2) Cyclidium vorax spec. nov. grouped with C. glaucoma and C. sinicum, which supports the assignment of the new species to the genus Cyclidium; (3) Cyclidium remains non-monophyletic with the addition of the new sequence.


Assuntos
Oligoimenóforos/classificação , Oligoimenóforos/genética , China , DNA de Protozoário/genética , Água Doce , Oligoimenóforos/citologia , Filogenia , Especificidade da Espécie
9.
J Parasitol ; 106(2): 295-307, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32316032

RESUMO

Cyclospora cayetanensis is a coccidian parasite of humans of known and growing importance. However, we are surprisingly naïve as to our understanding of how to diagnose it and how it develops inside the human body. Here we provide details of the developmental stages of C. cayetanensis in the gallbladder of a 33-yr-old male with human immunodeficiency virus. The gallbladder was removed surgically in 2001 because of severe abdominal pain. For the present study, the archived paraffin block of gallbladder was processed for light microscopy and transmission electron microscopy (TEM). Histological sections were examined after staining with hematoxylin and eosin (HE) or using the periodic acid Schiff (PAS) reaction. Immature and mature asexual stages, gamonts, and oocysts were seen in epithelial cells, both in the superficial epithelium and in glands. The merozoites were present singly, in pairs, and 3 or more in a single parasitophorous vacuole in the host cytoplasm. Up to 6 nuclei were seen in immature schizonts without evidence of merozoite formation. Mature schizonts were 7.6 × 5.1 µm and contained up to 10, 3-4 µm long merozoites. Merozoites were 0.6 to 2.0 µm wide, and their shape varied from pear-shaped to slender. Merozoites were generally PAS-positive; however, some were intensely positive, some had only minute granules, while others were PAS-negative. The microgamonts (male) were 6.6 × 5.2 µm and contained fewer than 20 microgametes around a residual body. The microgametes were up to 2 µm long and were flagellated. Macrogamonts (female) contained distinctive eosinophilic wall-forming bodies that varied in size and were less than 1 µm in HE-stained sections. Macrogamonts were 5.8-6.5 × 5.3-6.5 µm. Oocysts in sections were unsporulated and had a diameter of 5.7-7.5 µm. The TEM examination confirmed the histologic findings. The DNA extracted from paraffin sections was confirmed as C. cayetanensis with real-time PCR. The detailed description of the life cycle stages of C. cayetanensis reported here in an immunosuppressed patient could facilitate histopathologic diagnosis of this parasite. We have shown that the parasite's development more closely resembles that of Cystoisospora than Eimeria and that the parasite has multiple nuclei per immature meront indicating schizogony, and we have undermined evidence for a Type II meront.


Assuntos
Cyclospora/crescimento & desenvolvimento , Ciclosporíase/parasitologia , Vesícula Biliar/parasitologia , Infecções por HIV/complicações , Adulto , Cyclospora/genética , Cyclospora/ultraestrutura , Ciclosporíase/imunologia , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Feminino , Vesícula Biliar/patologia , Vesícula Biliar/ultraestrutura , Humanos , Hospedeiro Imunocomprometido , Estágios do Ciclo de Vida , Masculino , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real
10.
J Parasitol ; 106(2): 312-315, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32330280

RESUMO

The purpose of this study was to describe the prevalence and incidence of Neospora caninum infection in dogs that are in close contact with dairy cattle and to identify possible risk factors associated with the infection in this population. Twenty-four dogs located in 8 different dairy farms of Aguascalientes, Mexico, were evaluated for a 6-mo period. Once a month a sample of serum and a sample of peripheral blood was collected. The serum was used to detect antibodies against N. caninum by means of the indirect immunofluorescence technique, and the blood was used to detect parasite's DNA. The association between seroprevalence and possible risk factors was estimated using logistic regression. The prevalence of anti-N. caninum antibodies was 54% in the first month, 62% in the last month, and the incidence was 8.69%. One farm had no positive cases. Antibody titers ranged from 1:50 to 1:800. Parasite DNA was not detected in any of the samples. Only the age (>6 yr) of the dogs was identified as a risk factor for infection by N. caninum (P ≤ 0.05).


Assuntos
Coccidiose/veterinária , Doenças do Cão/parasitologia , Neospora , Fatores Etários , Animais , Anticorpos Antiprotozoários/sangue , Bovinos , Coccidiose/epidemiologia , Coccidiose/parasitologia , DNA de Protozoário/sangue , DNA de Protozoário/isolamento & purificação , Indústria de Laticínios , Doenças do Cão/epidemiologia , Cães , Feminino , Incidência , Masculino , México/epidemiologia , Neospora/genética , Neospora/imunologia , Prevalência , Fatores de Risco
11.
PLoS Negl Trop Dis ; 14(4): e0008175, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32267840

RESUMO

We recently developed a superhydrophobic cone-based method for the collection of mosquito excreta/feces (E/F) for the molecular xenomonitoring of vector-borne parasites showing higher throughput compared to the traditional approach. To test its field applicability, we used this platform to detect the presence of filarial and malaria parasites in two villages of Ghana and compared results to those for detection in mosquito carcasses and human blood. We compared the molecular detection of three parasites (Wuchereria bancrofti, Plasmodium falciparum and Mansonella perstans) in mosquito E/F, mosquito carcasses and human blood collected from the same households in two villages in the Savannah Region of the country. We successfully detected the parasite DNA in mosquito E/F from indoor resting mosquitoes, including W. bancrofti which had a very low community prevalence (2.5-3.8%). Detection in the E/F samples was concordant with detection in insect whole carcasses and human blood, and a parasite not vectored by mosquitoes was detected as well.Our approach to collect and test mosquito E/F successfully detected a variety of parasites at varying prevalence in the human population under field conditions, including a pathogen (M. perstans) which is not transmitted by mosquitoes. The method shows promise for further development and applicability for the early detection and surveillance of a variety of pathogens carried in human blood.


Assuntos
Culicidae/parasitologia , DNA de Protozoário/isolamento & purificação , Fezes/parasitologia , Malária/parasitologia , Mosquitos Vetores/parasitologia , Animais , DNA de Helmintos/genética , DNA de Protozoário/genética , Características da Família , Gana/epidemiologia , Humanos , Malária/epidemiologia , Técnicas de Diagnóstico Molecular/métodos , Plasmodium falciparum/genética , Prevalência , Wuchereria bancrofti/genética
12.
PLoS Negl Trop Dis ; 14(4): e0008148, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282820

RESUMO

BACKGROUND: Echinococcosis is a life-threatening parasitic disease caused by Echinococcus spp. tapeworms with over one million people affected globally at any time. The Echinococcus spp. tapeworms in the human body release DNA to the circulatory system, which can be a biomarker for echinococcosis. Cell-free DNA (cfDNA) is widely used in medical research and has been applied in various clinical settings. As for echinococcosis, several PCR-based tests had been trialed to detect cell-free Echinococcus spp. DNA in plasma or serum, but the sensitivity was about 20% to 25%. Low sensitivity of PCR-based methods might be related to our limited understanding of the features of cell-free Echinococcus spp. DNA in plasma, including its concentration, fragment pattern and release source. In this study, we applied ultra-high-throughput sequencing to comprehensively investigate the characteristics of cell-free Echinococcus spp. DNA in plasma of echinococcosis patients. METHODOLOGY/PRINCIPAL FINDINGS: We collected plasma samples from 23 echinococcosis patients. Total plasma cfDNA was extracted and sequenced with a high-throughput sequencing platform. An average of 282 million read pairs were obtained for each plasma sample. Sequencing data were analyzed with bioinformatics workflow combined with Echinococcus spp. sequence database. After identification of cell-free Echinococcus spp. reads, we found that the cell-free Echinococcus spp. reads accounted for 1.8e-5 to 4.0e-9 of the total clean reads. Comparing fragment length distribution of cfDNA between Echinococcus spp. and humans showed that cell-free Echinococcus spp. DNA of cystic echinococcosis (CE) had a broad length range, while that of alveolar echinococcosis (AE) had an obvious peak at about 135 bp. We found that most of the cell-free Echinococcus spp. DNA reads were from the nuclear genome with an even distribution, which might indicate a random release pattern of cell-free Echinococcus spp. DNA. CONCLUSIONS/SIGNIFICANCE: With ultra-high-throughput sequencing technology, we analyzed the concentration, fragment length, release source, and other characteristics of cell-free Echinococcus spp. DNA in the plasma of echinococcosis patients. A better understanding of the characteristics of cell-free Echinococcus spp. DNA in plasma may facilitate their future application as a biomarker for diagnosis.


Assuntos
DNA de Protozoário/sangue , Equinococose/diagnóstico , Equinococose/parasitologia , Echinococcus/genética , Echinococcus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adolescente , Adulto , Animais , Sequência de Bases , Biomarcadores , Criança , DNA de Protozoário/isolamento & purificação , Feminino , Genoma de Protozoário , Humanos , Masculino , Pessoa de Meia-Idade , Plasma , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA , Adulto Jovem
13.
PLoS Negl Trop Dis ; 14(4): e0008195, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32320399

RESUMO

BACKGROUND: Intestinal parasites such as Cryptosporidium spp., Giardia lamblia and Entamoeba histolytica can cause severe diarrhea, especially among children in developing countries. This study aims to determine the frequency of Cryptosporidium spp., Giardia lamblia and Entamoeba histolytica in children with diarrhea and identify risk factors for infection. METHODOLOGY: We conducted a cross-sectional study in children aged 0-168 months hospitalized with diarrhea in three regions of Mozambique, from June 2014 to January 2018. Following consent, caretakers were interviewed and a single stool specimen was collected from each child to diagnose Cryptosporidium spp., G. lamblia and E. histolytica using commercial immune-enzymatic assay (TechLab, Inc, Blacksburg, VA, USA). Anthropometric data were collected from the clinical reports. Multivariable logistic regression models were built to identify risk factors for Cryptosporidium spp. and G. lamblia infection. RESULTS: Twenty-one percent of all specimens (212/1008) presented at least one parasitic infection. Cryptosporidium spp. infection was the most common 12.0% (118/985), followed by G. lamblia 9.7% (95/983) and E. histolytica 2.0% (20/1004). Risk factors for infection by Cryptosporidium spp. were: provenience (children from Nampula province showed the highest risk, OR: 8.176; CI: 1.916-34.894; p-value < 0.01); animal contact (children with animal contact had a protective effect OR: 0.627; CI: 0.398-0.986; p-value < 0.05); underweight (children severely underweight showed a risk of 2.309; CI: 1.310-4.069; p-value < 0.05). Risk factors for infection by G. lamblia were: age (group with highest risk, 60-168 months (OR: 2.322; CI: 1.000-5.393, p-value > 0.05)); and living in a household with five or more members (OR: 2.141; CI: 1.286-3.565, p-value < 0.01). CONCLUSIONS: Parasitic infection is common among children with diarrhea. Routine testing, standard treatment, and assessment for risk exposure of children with diarrhea should be implemented at health facilities in Mozambique.


Assuntos
Criptosporidiose/epidemiologia , Diarreia/parasitologia , Entamebíase/epidemiologia , Giardíase/epidemiologia , Adolescente , Animais , Criança , Pré-Escolar , Estudos Transversais , Cryptosporidium/isolamento & purificação , DNA de Protozoário/análise , Diarreia/epidemiologia , Entamoeba histolytica/isolamento & purificação , Fezes/parasitologia , Feminino , Giardia lamblia/isolamento & purificação , Humanos , Lactente , Recém-Nascido , Modelos Logísticos , Masculino , Moçambique/epidemiologia , Análise Multivariada , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco
14.
PLoS Negl Trop Dis ; 14(4): e0008253, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324738

RESUMO

BACKGROUND: In the Mediterranean basin, Leishmania infantum is the causative agent of visceral leishmaniasis (VL), a zoonosis in which the dog is the primary domestic reservoir, although wildlife may have a leading role in the sylvatic cycle of the disease in some areas. Infections without disease are very frequent. There is limited information regarding the role that VL patients and asymptomatic infected individuals could be playing in the transmission of L. infantum. Xenodiagnosis of leishmaniasis has been used in this descriptive study to explore the role of symptomatic and asymptomatic infected individuals as reservoirs in a recent focus of leishmaniasis in southwestern Madrid, Spain. METHODOLOGY AND MAIN FINDINGS: Asymptomatic blood donors (n = 24), immunocompetent patients who were untreated (n = 12) or treated (n = 11) for visceral leishmaniasis (VL), and immunocompromised patients with VL (n = 3) were enrolled in the study. Their infectivity to Phlebotomus perniciosus was studied by indirect xenodiagnosis on peripheral blood samples. Quantitative polymerase chain reaction of blood samples from immunocompetent patients untreated for VL and immunocompromised untreated, treated and under secondary prophylaxis for VL was performed. Antibodies against Leishmania were studied by indirect fluorescent antibody and rK39-immunochromatographic tests. A lymphoproliferative assay with a soluble Leishmania antigen was used to screen for leishmaniasis infection in the healthy population. Sixty-two xenodiagnostic tests were carried out and 5,080 sand flies were dissected. Positive xenodiagnosis was recorded in four patients, with different sand fly infection rates: 1 immunosuppressed HIV / L. infantum coinfected asymptomatic patient, 1 immunosuppressed patient with multiple myeloma and symptomatic active VL, and 2 immunocompetent patients with untreated active VL. All blood donors were negative for both xenodiagnosis and conventional PCR. CONCLUSIONS / SIGNIFICANCE: There is no consensus amongst authors on the definition of an 'asymptomatic case' nor on the tools for screening; we, therefore, have adopted one for the sake of clarity. Immunocompetent subjects, both infected asymptomatics and those treated for VL, are limited in number and appear to have no epidemiological relevance. The impact is limited for immunocompetent patients with untreated active VL, whilst immunosuppressed individuals undergoing immunosuppressive therapy and immunosuppressed individuals HIV / L. infantum coinfected were the most infectious towards sand flies. It is noteworthy that the HIV / L. infantum coinfected patient with asymptomatic leishmaniasis was easily infectious to sand flies for a long time, despite being under continuous prophylaxis for leishmaniasis. Accordingly, screening for latent Leishmania infection in HIV-infected patients is recommended in scenarios where transmission occurs. In addition, screening for VL in HIV-infected patients who have spent time in VL-endemic areas should also be implemented in non-endemic areas. More research is needed to better understand if some asymptomatic coinfected individuals contribute to transmission as 'super-spreaders'.


Assuntos
Reservatórios de Doenças , Transmissão de Doença Infecciosa , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/transmissão , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Doenças Assintomáticas/epidemiologia , DNA de Protozoário/sangue , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Psychodidae/parasitologia , Espanha/epidemiologia
15.
Zootaxa ; 4732(3): zootaxa.4732.3.6, 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32230251

RESUMO

The morphologies of the three freshwater stentorid ciliates in Korea, Stentor coeruleus (Pallas, 1766); Stentor muelleri Ehrenberg, 1831, and Stentor tartari Murthy Bai, 1974, were investigated based on live observations and protargol impregnation. The Korean population of S. tartari exhibits the following characteristics: body size 200-355 × 85-135 µm in vivo, 62-106 somatic kineties, 8-13 peristomial kineties, 110-180 adoral membranelles, mostly two macronuclear nodules and 5-18 micronuclei, reddish and colorless cortical granules and the presence of symbiotic algae. We identified S. tartari based on unique characteristics compared to close congeners. Korean populations of S. coeruleus and S. muelleri are congruent with previously described populations in most aspects of their morphologies. Here, for the first time, we report molecular gene sequence information for S. tartari. Small subunit (SSU) rRNA gene sequence-based phylogeny indicates that S. tartari, which has multiple macronuclei, forms a monophyletic group with other Stentor species having a single macronucleus. Our findings based on morphology and SSU rRNA gene sequence information corroborate the hypothesis that the elongated macronucleus evolved from the compact single or multi macronucleus state.


Assuntos
Cilióforos , Animais , China , DNA de Protozoário , Filogenia , República da Coreia
16.
Parasit Vectors ; 13(1): 186, 2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-32272968

RESUMO

BACKGROUND: From a veterinary-medical point of view, the stable fly, Stomoxys calcitrans, is perhaps the economically most important blood-sucking muscoid fly species (Diptera: Muscidae), owing to its worldwide occurrence, frequently high local abundance, direct harm caused to livestock, pet animals and humans, as well as its vector role. Considering the latter in the context of protozoan parasites, the stable fly is a mechanical vector of trypanosomes and Besnoitia besnoiti. However, its role as a vector of piroplasms appears to be seldom studied, despite old data suggesting mechanical transmission of babesiae by dipteran flies. METHODS: In this study 395 stable flies (and one Haematobia stimulans) were collected at a cattle farm with known history of bovine theileriosis, and at further nine, randomly chosen locations in Hungary. These flies were separated according to sex (30 of them also cut into two parts: the head with mouthparts and the thorax-abdomen), followed by individual DNA extraction, then screening for piroplasms by PCR and sequencing. RESULTS: In stable flies, Theileria orientalis and T. capreoli were identified at the cattle farm and T. equi was identified in three other locations. At the cattle farm, significantly more male stable flies carried piroplasm DNA than females. There was no significant difference between the ratio of PCR-positive flies between the stable (void of cattle for at least two hours) and the pen on the pasture with cattle at the time of sampling. Among dissected flies (29 S. calcitrans and 1 H. stimulans), exclusively the thoracic-abdominal parts were PCR-positive, whereas the head and mouthparts remained negative. CONCLUSIONS: Theileria DNA is detectable in stable flies, in the case of T. orientalis at least for two hours after blood-feeding, and in the case of T. capreoli also in the absence of infected hosts (i.e. roe deer). Male flies rather than females, and thoracic-abdominal (most likely crop) contents rather than mouthparts may pose a risk of mechanical transmission. These data suggest that it is worth to study further the vector role of stable flies in the epidemiology of theilerioses, in which not the immediate, but rather the delayed type transmission seems possible.


Assuntos
Insetos Vetores/parasitologia , Muscidae/parasitologia , Theileria/isolamento & purificação , Theileriose/epidemiologia , Animais , Bovinos , Doenças dos Bovinos/parasitologia , DNA de Protozoário , Cervos/parasitologia , Feminino , Hungria , Intestinos/parasitologia , Masculino , Patologia Molecular/métodos , RNA Ribossômico 18S
17.
Parasit Vectors ; 13(1): 200, 2020 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-32306993

RESUMO

BACKGROUND: Approximately 30% of children worldwide are infected with gastrointestinal parasites. Depending on the species, parasites can disrupt intestinal bacterial microbiota affecting essential vitamin biosynthesis. METHODS: Stool samples were collected from 37 asymptomatic children from a previous cross-sectional Argentinian study. A multi-parallel real-time quantitative PCR was implemented for Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, Cryptosporidium spp., Entamoeba histolytica and Giardia duodenalis. In addition, whole-genome sequencing analysis was conducted for bacterial microbiota on all samples and analyzed using Livermore Metagenomic Analysis Toolkit and DIAMOND software. Separate analyses were carried out for uninfected, Giardia-only, Giardia + helminth co-infections, and helminth-only groups. RESULTS: For Giardia-only infected children compared to uninfected children, DNA sequencing data showed a decrease in microbiota biodiversity that correlated with increasing Giardia burden and was statistically significant using Shannon's alpha diversity (Giardia-only > 1 fg/µl 2.346; non-infected group 3.253, P = 0.0317). An increase in diversity was observed for helminth-only infections with a decrease in diversity for Giardia + helminth co-infections (P = 0.00178). In Giardia-only infections, microbiome taxonomy changed from Firmicutes towards increasing proportions of Prevotella, with the degree of change related to the intensity of infection compared to uninfected (P = 0.0317). The abundance of Prevotella bacteria was decreased in the helminths-only group but increased for Giardia + helminth co-infections (P = 0.0262). Metagenomic analysis determined cobalamin synthesis was decreased in the Giardia > 1 fg/µl group compared to both the Giardia < 1 fg/µl and the uninfected group (P = 0.0369). Giardia + helminth group also had a decrease in cobalamin CbiM genes from helminth-only infections (P = 0.000754). CONCLUSION: The study results may provide evidence for an effect of parasitic infections enabling the permissive growth of anaerobic bacteria such as Prevotella, suggesting an altered capacity of vitamin B12 (cobalamin) biosynthesis and potential impact on growth and development in children .


Assuntos
Coinfecção , Microbioma Gastrointestinal/genética , Intestinos , Parasitos/genética , Vitamina B 12/genética , Animais , Criança , Pré-Escolar , Coinfecção/microbiologia , Coinfecção/parasitologia , Estudos Transversais , DNA de Helmintos , DNA de Protozoário , Feminino , Genes Bacterianos , Giardia lamblia/classificação , Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Helmintos/classificação , Helmintos/genética , Helmintos/isolamento & purificação , Humanos , Intestinos/microbiologia , Intestinos/parasitologia , Masculino , Metagenômica , Parasitos/classificação , Parasitos/isolamento & purificação , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real , Vitamina B 12/metabolismo , Sequenciamento Completo do Genoma
18.
Rev Soc Bras Med Trop ; 53: e20190433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348430

RESUMO

INTRODUCTION: As highly specific molecular biology-based techniques may not be sensitive enough for the diagnosis of American tegumentary leishmaniasis (ATL), clinicians frequently rely on immunological tests before treatment initiation. Hence, the correct combination of diagnostic tests is imperative for ATL diagnosis. We aimed to evaluate the accuracy of the Montenegro (Leishmanin) skin test (MST) in polymerase chain reaction (PCR)-negative patients to accurately detect ATL. METHODS: Patients with a clinical picture compatible with ATL were divided into ATL (confirmed by lesion smear, culture indirect immunofluorescence, and/or histopathology) and no-ATL (diseases that can mimic leishmaniasis) groups. Conventional PCR for the minicircle kDNA of Leishmania was performed, and the MST was carried out for PCR-negative patients. RESULTS: Ninety-nine patients were included in this study, including 79 diagnosed with ATL (6 with mucocutaneous leishmaniasis) and 20 without ATL (no-ATL group). The MST showed a high sensitivity of 90.0% (95% confidence interval [CI] = 69.90-97.21) in PCR-negative patients that was 10% higher than the sensitivity reported in PCR-positive population (79.66%; 95% CI = 67.73-87.96). CONCLUSIONS: One of the most important reasons for PCR negativity among patients with active ATL is the presence of a strong cellular immunological response, especially in chronic and mucocutaneous leishmaniasis. This reinforces the considerable utility of the tests that detect cellular responses against Leishmania antigens such as the MST in PCR-negative patients when the performance in screening situations is questionable.


Assuntos
Testes Intradérmicos/métodos , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Adulto , Idoso , Doença Crônica , Estudos Transversais , DNA de Protozoário/genética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Leishmania braziliensis/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade
19.
Ann Parasitol ; 66(1): 19­25, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32198992

RESUMO

Blastocystis sp. is one of the most prevalent human parasites with a vast variety of non-human hosts. The aim of the present study was to determine the subtype distribution of Blastocystis in humans and trace the route of transmission by molecular data and phylogenetic analysis. Stool samples were collected from patients who referred to 14 medical laboratories in Kurdistan, Iran. All the samples were examined using the direct wet mount and formalinether concentration techniques. DNA extraction was carried out for 30 microscopically positive isolates and 33 negative samples. DNA amplification and subtype identification were also performed using the barcoding method and sequencing techniques. Of 1383 stool samples, 239 (17.3%) were infected with Blastocystis sp. Out of the 24 sequenced isolates, two (8.3%), six (25%), and 16 (66.6 %) belonged to the ST1, ST2, and ST3 subtypes, respectively. Bioinformatics analysis indicated that all the isolates were genetically similar to animal isolates. Blastocystis sp. was very common and ST1, ST2, and ST3 subtypes were prevalent in the study population. Bioinformatics analysis suggests that zoonotic transmission plays an important role in Blastocystis sp. distribution in Kurdistan province.


Assuntos
Infecções por Blastocystis , Blastocystis , Filogenia , Zoonoses , Animais , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/transmissão , DNA de Protozoário/genética , Fezes/parasitologia , Variação Genética , Humanos , Irã (Geográfico) , Zoonoses/parasitologia , Zoonoses/transmissão
20.
Ann Parasitol ; 66(1): 111­114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32199003

RESUMO

Acanthamoeba spp. are ubiquitous in both natural and man-made environments and have been isolated in lakes, recreational pools, tap water, and air conditioning systems. Twenty surface water (SW) samples were collected from different sampling areas of Lake Buhi. Water samples were pelleted, cultured in NNA lawned with Escherichia coli and observed microscopically. 10% of samples (2/20) were positive for amoebic growth and were furthered tested using molecular techniques. Polymerase chain reaction showed the presence of Acanthamoeba sp. DNA. The presence of potentially pathogenic Acanthamoeba sp. poses a public health concern. The formulation of policies for proper information dissemination and control measures to avert the contraction of pathogenic FLA as well as other WBPP should be seriously considered.


Assuntos
Acanthamoeba , Lagos , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , DNA de Protozoário/genética , Lagos/parasitologia , Filipinas , Reação em Cadeia da Polimerase
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